SUMMARY There are fundamental gaps in our understanding of how cells tolerate genotoxicity and intrinsic DNA replication stress while accumulating the mutations that incite and fuel carcinogenesis. Unfortunately, the DNA damage tolerance and mutability acquired during carcinogenesis also allow cancer cells to resist chemotherapy. Thus, current gaps in our knowledge of DNA damage tolerance and mutagenesis limit our understanding of carcinogenesis and preclude effective prevention and treatment of cancer. Our long-term goal is to solve the related problems of how neoplastic cells tolerate DNA damage and replication stress while mutating their genomes during carcinogenesis. The objective here, a critical step in the pursuit of that goal, is to mechanistically define a novel cancer cell-specific DNA damage-tolerance and error-prone DNA synthesis pathway and determine its role in carcinogenesis. Remarkably, we discovered that Melanoma Antigen-A4 (MAGE-A4, a Cancer/Testes Antigen or 'CTA') is an activating binding partner of the DNA repair protein RAD18 (an E3 ubiquitin ligase) in cancer cells. MAGE-A4 is absent from normal somatic cells and aberrantly expressed at very high levels in cancer cells. MAGE-A4 expression is also associated with poor patient prognosis, yet its potential contribution to tumorigenesis is untested. Based on exciting and compelling preliminary studies, we will test the central hypothesis that MAGE-A4 binds RAD18 to reprogram ubiquitin signaling, leading to pathological Trans-Lesion Synthesis (TLS), DNA damage-tolerance and mutability that drive carcinogenesis. The rationale is that defining the contribution of MAGE-A4 to tumorigenesis will allow therapeutic strategies that specifically target vulnerabilities of neoplastic cells. The Specific Aims are: (1) Define molecular mechanisms of MAGE-A4/RAD18-mediated DNA damage tolerance and mutagenesis. (2) Determine impact of MAGE-A4/Rad18 on DNA damage-sensitivity, mutagenesis and carcinogenesis in vivo. (3) Determine contribution of MAGE-A4/Rad18 to tolerance of oncogenic stress. In support of SA1 we will determine the mechanism(s) by which MAGE-A4 influences error-free and error-prone (mutagenic) replication of damaged and undamaged DNA templates. For SA2 we will use novel mouse models (already in hand) to determine how conditional MAGE-A4 expression impacts DNA damage tolerance, genome stability and chemical carcinogenesis in vivo. For SA3 we will determine how MAGE-A4/RAD18 signaling affects tolerance of oncogene-induced replication stress (in cultured cells) and oncogene-induced tumorigenesis (in vivo). The proposed ideas and research are innovative because nobody has ever tested how CTAs affect genome maintenance, carcinogenesis or cancer therapy. The proposed work is significant because we will provide new paradigms for genome maintenance that are relevant to environmental exposures, mutagenesis, tumorigenesis and cancer therapy in humans. This work may lead to novel strategies for targeting DNA damage tolerance and mutability specifically in cancer cells, thereby enhancing the efficacy and selectivity of chemotherapy.